Methods for Altering MRNA Splicing and Treating Familial Dysautonomia and Other Mechanistically Related Disorders

ABSTRACT

This invention relates to methods for altering the splicing of mRNA in cells. In particular, this invention also relates to methods for increasing the ratio of wild type to misspliced forms of mRNA and corresponding encoded proteins in cells possessing a mutant gene encoding either the i) misspliced mRNA corresponding to the mutant protein or ii) a component in the splicing machinery responsible for processing the misspliced mRNA. In addition, this invention relates to treating individuals having a disorder associated with a misspliced mRNA, such as Familial Dysautonomia or Neurofibromatosis 1, by administering to such an individual a cytokinin such as kinetin.

This application is a divisional of application Ser. No. 10/956,601,filed Oct. 1, 2004, which claims priority to U.S. provisionalapplication Ser. No. 60/508,465, filed Oct. 3, 2003 and U.S. provisionalapplication Ser. No. 60/536,287, filed Jan. 13, 2004, the contents ofeach of which are incorporated in their entirety.

This invention was made with United States Government support undergrants from the National Institutes of Neurological Disorders andStroke. The United States Government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates to methods for altering the splicing of mRNA incells. In addition, this invention also relates to methods forcorrecting the ratio of wild type to mutant spliced forms of mRNA andcorresponding encoded proteins in cells possessing a mutant geneencoding either i) the misspliced mRNA corresponding to the mutantprotein or ii) a component of the splicing machinery. In addition, thisinvention relates to treating individuals having a disorder associatedwith a misspliced mRNA, such as familial dysautonomia, by administeringto such an individual a cytokinin such as kinetin.

In particular, the invention relates to enhancing correct mRNA splicingin order to increase cellular levels of normal or wild type IKAP mRNA orprotein encoded by a IKBKAP gene in various cell types. The defectivesplicing of pre-mRNA is a major cause of human disease.

BACKGROUND OF THE INVENTION

Exon skipping is a common result of splice mutations and has beenreported in a wide variety of genetic disorders¹, yet the underlyingmechanism is poorly understood. Often, such mutations are incompletelypenetrant, and low levels of normal transcript and protein aremaintained¹. Familial dysautonomia (FD) (MIM#2239001), also known asRiley Day syndrome or hereditary sensory and autonomic neuropathy III(HSAN-III), is the best-known and most common member of a group ofcongenital sensory and autonomic neuropathies (HSAN) characterized bywidespread sensory and variable autonomic dysfunction (Axelrod F B:(1996) Autonomic and Sensory Disorders. In: Principles and Practice ofMedical Genetics, 3rd edition, A E H Emory and D L Rimoin eds. ChurchillLivingstone, Edinburgh. pp 397-411; Axelrod F B (2002) HereditarySensory and Autonomic Neuropathies: Familial Dysautonomia and otherHSANs. Clin Auton Res 12 Supplement 1, 2-14). FD affects neuronaldevelopment and is associated with progressive neuronal degeneration.Multiple systems are impacted resulting in a markedly reduced quality oflife and premature death (Axelrod F B: (1996) Autonomic and SensoryDisorders. In: Principles and Practice of Medical Genetics, 3rd edition,A E H Emory and D L Rimoin eds. Churchill Livingstone, Edinburgh. pp397-411; Axelrod F B (2002) Hereditary Sensory and AutonomicNeuropathies: Familial Dysautonomia and other HSANs. Clin Auton Res 12Supplement 1, 2-14).

FD is a recessive disorder that has a remarkably high carrier frequencyof 1 in 30 in the Ashkenazi Jewish population⁵. FD is caused bymutations in the IKBKAP gene^(2,3) (Genbank Accession No.NM_(—)003640.), and all cases described to date involve an intron 20mutation that results in a unique pattern of tissue-specific exonskipping. Accurate splicing of the mutant IKBKAP allele is particularlyinefficient in the nervous system. Three FD mutations have beenidentified in the I-k-B kinase (IKK) complex-associated protein(IKBKAP): IVS20^(+6T→C), which leads to variable, tissue-specificskipping of exon 20 (FIG. 1 a), R696P, and P914L^(2,3,6). All FDpatients tested to date carry at least one IVS20^(+6T→C) mutation, withmore than 99.5% being homozygous, and the remainder being heterozygouswith either R696P or P914L on the alternate allele.

The IVS20^(+6T→C) mutation does not cause complete loss of function.Instead, it results in a tissue-specific decrease in splicing efficiencyof the IKBKAP transcript; cells from patients retain some capacity toproduce normal mRNA and IKAP protein (Slaugenhaupt et al. (2001)Tissue-specific expression of a splicing mutation in the IKBKAP genecauses familial dysautonomia. Am J Hum Genetics 68:598-605). The mRNA iswidely distributed. Highest levels are in the nervous system, butsubstantial amounts are also present in peripheral organs (Mezey et al.(2003) Of splice and men: what does the distribution of IKAP mRNA in therat tell us about the pathogenesis of familial dysautonomia? BrainResearch 983:209). It has been reported previously that all FD tissuestested express both wild-type (WT) and mutant (MU) IKBKAP mRNA^(2,4).The effect of the most common (splicing) mutation varies from tissue totissue—neuronal tissues seem primarily to express mutant mRNA; somatictissues express roughly equal levels of normal and mutant mRNA. Accuratemeasurement of the ratio of the two mRNA species using both densitometryand real-time quantitative PCR has revealed that the levels of WT IKBKAPmRNA vary between tissues and are lowest in central and peripheralnervous systems⁴. This leads to a drastic reduction in the amount ofIKAP protein in these tissues.

There are other disorders that are caused, at least in part, bymissplicing including Neurofibromatosis 1 (NF1), also known as vonRecklinghausen NF or Peripheral NF. NF1 occurs in 1:4,000 births and ischaracterized by multiple cafe-au-lait spots and neurofibromas on orunder the skin. Enlargement and deformation of bones and curvature ofthe spine may also occur (Riccardi, 1992, Neurofibromatosis: phenotype,natural history, and pathogenesis. 2nd ed. Baltimore: Johns HopkinsUniversity Press). Occasionally, tumors may develop in the brain, oncranial nerves, or on the spinal cord. About 50% of people with NF alsohave learning disabilities (Chapter 6 in Rubenstein and Korf, 1990,Neurofibromatosis: a handbook for patients, families, and health-careprofessionals. New York: Thieme Medical Publishers).

The NF1 gene was identified and the protein product characterized in1990 (Cawthon et al., 1990, Cell 62: 193-201; Wallace et al., 1990,Science 249:181-6). The entire sequence of the expressed NF1 gene hasbeen reported (Viskochil et al., 1993, Annu Rev Neurosci 16: 183-205;Gutmann and Collins, 1993, Neuron 10: 335-43; Genbank Accession No.NM_(—)000267). The gene is has at least 59 exons and codes for a 2818amino acid protein called neurofibromin. To date, 180 different NF1mutations have been identified. The NF1 Genetic Analysis Consortiummaintains a database of mutations identified in more than 45collaborating laboratories throughout the world. According to data fromthe Consortium, the NF1 mutations described to date include 4chromosomal rearrangements, 89 deletions (14 deletions involving theentire gene, 35 deletions involving multiple exons, and 37 smalldeletions), 23 insertions (3 large and 20 small), 45 point mutations (29stop mutations and 16 amino acid substitutions), and 18 intronicmutations affecting splicing, and 4 mutations in 3′ untranslated regionof the gene. About 30% of NF1 patients carry a splice mutation resultingin the production of one or several shortened transcripts (Vandenbrouckeet al., 2002, BMC Genomics 3:13 and Serra et al., 2001, Hum Genet.108:416-29).

Cytokinins are a class of plant hormones defined by their ability topromote cell division in plant tissue explants in the presence of anauxin, such as indoleacetic acid, and nutrients, including vitamins,mineral salts, and sugar. In promoting cell division of plant cells,cytokinins are active at low concentrations (as low 0.01 parts permillion (ppm)), but exhibit activity only in the presence of an auxin.Certain cytokinins, including zeatin and6-(3,3-dimethylallyl)-aminopurine, also occur as the base moietycomponents of transfer RNA in yeast, bacterial, animal cells and plantcells. The cytokinin kinetin (6-furfuryl-aminopurine) forms complexeswith certain RNA-binding proteins of wheat embryo extracts and appearsto promote protein synthesis in plants (see, e.g., Spirin and Ajtkhozhin(1985) Trends in Biochem. Sci., p. 162). Kinetin and other cytokininsare used in conjunction with auxin used in horticulture and in planttissue culture, such as in the production of plantlets from plant callustissue. Cytokinins are also used in the production of protein-rich yeast(see e.g., East German Patent No. 148,889 (1981) (Derwent World PatentIndex Abstract)) and to augment the growth of microbial cultures (MerckIndex, 10th Ed. (1983) Entry 5148, Merck and Co., Rahway, N.J., U.S.A.).

Kinetin belongs to the family of N⁶-substituted adenine derivativesknown as cytokinins, or plant growth factors, that also includes zeatin,benzyladenine and 2iP (FIG. 1 b). Kinetin is also known as6-furfurylaminpurine (C₁₀H₉N₅) and has a molecular weight of 215.21(Soriano-Garcia and Parthsarathy,1975, Biochem Biophys Res Commun64:1062-8). Kinetin is currently marketed as an anti-aging ingredient inskin treatments due to its ability to ameliorate aging characteristicsin cultured human fibroblasts⁸, possibly through anti-oxidant activity⁹.

Certain cytokinins have been shown to inhibit the growth of tumor cellsin vitro (see, e.g., Katsaros et al. (1987) FEBS Lttrs. 223:97-103). Itappears that this effect is mediated via the cytotoxic affects ofadenosine analogs, such as the 6-(substituted amino) purine cytokinins,that interfere with tRNA methylating enzymes (Wainfan et al, (1973)Biochem. Pharmacol. 22:493-500). When immortalized fibroblast cells arecontacted with adenosine analogs the cultured cells exhibit decreasedgrowth rate and a change in morphology from the normal flattenedelongated morphology typical of cultured fibroblasts to a very elongatedspindle-shape characteristic of a cytotoxic response. The very elongatedshape of immortalized cells exhibiting this response is not shapecharacteristic of young, healthy, primary cultures of normal diploidfibroblasts.

Kinetin has been shown to be capable of delaying or preventing a host ofage-related changes of human skin fibroblasts grown in laboratoryculture which has led to its incorporation into topical skin products.West MID (1994) The cellular and molecular biology of skin aging. ArchDermatol 130:87-95. Fibroblasts, which produce collagen and elastin,have been shown to decrease in number and vitality as skin ages not onlyin vitro, but also in vivo. The number of fibroblasts decreases at least50% between birth and the age of 80 years. West MID (1994) The cellularand molecular biology of skin aging. Arch Dermatol 130:87-95. Rattan SI, Clark B F. (1994) Kinetin delays the onset of aging characteristicsin human fibroblasts. Biochem Biophys Res Commun 201:665-72. Kinetin hasbeen shown to delay or prevent a range of cellular changes associatedwith in vitro aging of human skin cells, including alterations in cellmorphology, growth rate, size, cytoskeletal organization, macromolecularsynthetic activity and accumulation of lipofuscin aging pigments butkinetin did not alter the maximum in vitro life span of human skin cellsor their ability to multiply in culture. Rattan S I, Clark B F. (1994)Kinetin delays the onset of aging characteristics in human fibroblasts.Biochem Biophys Res Commun 201:665-72. Thus, kinetin was devoid ofactivities associated with cellular immortalization, malignanttransformation and carcinogenesis. Rattan S I, Clark B F. (1994) Kinetindelays the onset of aging characteristics in human fibroblasts. BiochemBiophys Res Commun 201:665-72.

Using rats as a mammalian model, it has been shown that plant cytokininscan affect lipid peroxidation in erythrocyte, muscle, liver, heart andkidney tissue, Celik I, Tuluce Y, Ozok N.(2002) Effects of indoleaceticacid and kinetin on lipid peroxidation levels in various rat tissues.Turk J Biol 26;193-196. Celik et al.(2002) showed that kinetin had muchless toxicity as compared to indoleacetic acid (IAA), when administeredorally to rats. Celik I, Tuluce Y, Ozok N.(2002) Effects of indoleaceticacid and kinetin on lipid peroxidation levels in various rat tissues.Turk J Biol 26;193-196. It was shown that IAA interacts primarily withthe liver and kidney tissue cells, resulting in lipid peroxidationsynthesis, whereas kinetin had no such effect in the liver or kidney.

SUMMARY OF THE INVENTION

This invention relates to methods and compositions for increasing theamount of wild type protein encoded by cells possessing a missplicedmRNA due to either a mutation in i) the misspliced mRNA correspondingmutant protein or ii) a component of the splicing machinery. Inpreferred embodiments, the misspliced mRNA is a mutant mRNA. Theincreased wild type protein results from contacting cells with oradministering to individuals one or more compounds which increases theamount of properly spliced mRNA. In preferred embodiments, the compoundis a cytokinin. In a more preferred embodiment, the cytokinin iskinetin, (6-furfurylaminopurine).

In one embodiment of this invention, cells possessing a mutant generesulting in misspliced mRNA are contacted with a cytokinin whichresults in an enhanced ratio of correctly spliced mRNA compared tomisspliced mRNA.

In another embodiment of this invention, cells or individuals arecontacted with a cytokinin (preferably kinetin) to treat neuronaldegeneration.

In another embodiment of this invention a cytokinin (preferably kinetin)is administered to an individual with a disorder or disease associatedwith a mutation resulting in misspliced mRNA.

In a preferred embodiment, individuals with familial dysautonomia (FD)are treated with a therapeutically effective amount of a cytokinin(preferably kinetin) to decrease missplicing of the IKBKAP transcriptand increase the ratio of wild type IKAP protein versus mutant IKAPprotein. In a specific embodiment, a pharmaceutical compositioncomprises the therapeutically effective amount of the cytokinin and isadministered to a subject suffering or likely to suffer from FD.

In another preferred embodiment, individuals with Neurofibromatosis 1(NF1) caused by missplicing are treated with a therapeutically effectiveamount of a cytokinin (preferably kinetin) to decrease missplicing ofthe NF1 transcript and increase the ratio of wild type neurofibrominprotein versus mutant neurofibromin protein. In a specific embodiment, apharmaceutical composition comprises the therapeutically effectiveamount of the cytokinin and is administered to a subject suffering orlikely to suffer from NF1.

The methods of the invention encompass in vivo and in vitro screeningassays to identify compounds that alter the splicing of misspliced mRNAtranscripts. In one embodiment, the misspliced mRNA transcript is amutant IKBKAP mRNA transcript. In a more specific embodiment, the mutantIKBKAP mRNA transcript carries a mutation that is present in a mutantIKBKAP mRNA in a subject with FD. In an even more specific embodiment,the mutant IKBKAP mRNA transcript has the IVS20^(+6T→C) mutation. Inanother another embodiment, the misspliced mRNA transcript is a mutantNF1 mRNA transcript. In a more specific embodiment, the mutant NF1 mRNAtranscript carries a mutation that is present in a mutant NF1 mRNA in asubject with NF1. Candidate compounds are screened for the ability toalter the splicing of misspliced mRNA transcripts comprising contactinga mammalian cell comprising DNA which comprises the gene (or a fragmentor variant thereof) which is misspliced with a candidate compound anddetermining the amount of misspliced and/or wild type mRNA transctipt.In a specific embodiment, candidate compounds are screened for theability to alter the inclusion of exon 20 of a wild type or mutatedIKBKAP gene. In more specific embodiments, the ratio of exon 20inclusion to exon 20 skipping is determined for spliced IKBKAP mRNAtranscripts in the presence and absence of the candidate compound. Analteration in the ratio of exon 20 skipping to exon 20 inclusionindicates that the candidate compound did alter the splicing of themisspliced mRNA.

It is an object of the present invention to provide a compound orcompounds which are suitable as therapeutic agent(s) for the treatmentof disorders involving missplicing of mRNAs, especially FD and NF1. Afurther object of the present invention is to provide a process andcompositions which are suitable for altering the splicing of mRNA in amammalian cell.

The present invention provides a composition which is capable ofaffecting mRNA splicing. In a preferred embodiment, the composition iscapable of altering mRNA splicing of the IKBKAP gene. In a specificpreferred embodiment, the composition is capable of altering IKBKAP genesplicing by increasing the inclusion of exon 20. In another preferredembodiment, the composition is capable of altering mRNA splicing of theNF1 gene. As such the composition is also useful as a pharmaceuticalcomposition to prevent, manage, and/or treat FD and/or NF1 caused bymissplicing according to the methods of the invention.

In some embodiments, the one or more cytokinins used in the methods ofthe invention are administered to a subject in need thereof incombination with at least one other compound that provides a therapeuticeffect. Examples of such other compounds include, but are not limitedto, antioxidants (such as (−)-epigallocatechin gallate) and tocotrienols(such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, andδ-tocotrienol).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Kinetin significantly increases production of wild-type IKBKAPtranscript in FD cells. (a) Schematic diagram illustrating the locationof the IVS20^(+6→C) mutation in IKBKAP and the two IKBKAP isoformsproduced from this allele in FD patients. (b) Chemical structures of thecytokinins tested in this study: kinetin (6-furfurylaminopurine),benzyladenine (6-benzylaminopurine), 2i P(6-(γ,γ-dimethyallylamino)purine), and zeatin(6-(4-hydroxy-3-methylbut-2-enylamino)purine). (c) RT-PCR analysis of 2independent FD lymphoblast cell lines, tested in triplicate, followingtreatment with 10 μM kinetin in 0.1% DMSO or DMSO alone. Previousexperiments demonstrated that DMSO has no effect on IKBKAP splicing.WT:MU ratios were determined using the integrated density value (IDV)obtained for each band and are shown beneath each lane. The sizes of theWT (including exon 20) and MU (excluding exon 20) PCR products areindicated on the right. Primer sequences and PCR conditions have beenpreviously described⁴.

FIG. 2 The action of kinetin on IKBKAP splicing is dose and timedependent and results in an increase of IKAP protein in FD lymphoblastcell lines. (a) RT-PCR demonstrating increasing WT:MU IKBKAP ratios as aresult of increased kinetin concentration. Sizes of the WT and MU bandsare shown on the right, and the IDV ratios are shown beneath each lane.(b) Western blot probed with an IKAP monoclonal antibody showing thatincreasing concentrations of kinetin result in an increase in IKAPprotein production in FD cells. The lower panel shows the same blotprobed with an IDE antibody as a protein loading control. (c) Graph ofWT:MU IKBKAP ratios at increasing kinetin concentrations determined byQPCR of FD cells (treated with kinetin in water in duplicate and eachamplified in triplicate) showing that higher doses of kinetin continueto increase WT:MU IKBKAP. Examination of panel (a) shows that at kinetinconcentrations of 100 μM the MU band is barely discernable usingdensitometry, therefore QPCR was used for this study. (d) Graph of WT:MUratios generated using IDV values following culturing of FD cells in 50μM kinetin for increasing lengths of time. All treatments were performedin duplicate and average data points are plotted.

FIG. 3 Kinetin has no effect on incorporation of exon 31 in thealternatively spliced MYO5A gene (a) schematic diagram illustrating twoof the alternative transcripts produced by MYO5A. The primers used foramplification are illustrated by arrows indicated on each isoform. (b)representative example of RT-PCR from FD lymphoblast cells showing nochange in the ratio of MYO5A isoforms by kinetin treatment. Nineindependent cell lines were tested.

FIG. 4 Kinetin increases WT:MU IKBKAP transcript ratio in the presenceor absence of nonsense mediated decay (NMD) of the mutant transcript.Two FD lymphoblast cell lines, FD1 and FD2, were untreated (U) ortreated with 50 pg/ml cycloheximide (C) to inhibit NMD, 100 μM kinetin(K), or cycloheximide+kinetin (C+K). RT-PCR amplification andfractionation of the amplified product on a 1.5% agarose gel wasperformed on cell extracts. WT:MU IKBKAP transcript ratios weredetermined using the integrated density value (IDV) obtained for eachband and are shown beneath each lane. Primer sequences and PCRconditions have been previously described ⁴

FIG. 5 Kinetin enhances inclusion of exon 20 in both the MU and WTIKBKAP minigene. (a) schematic diagram illustrating the minigeneconstructs and the location of the IVS20^(+6T→C) mutation. Vectorspecific primers used for RT-PCR analysis are shown. (b) RT-PCR of MUand WT minigene RNA isolated from HEK293 cells following transfectionand treatment with kinetin. PCR was performed using the primers T7 andBGH-R. Trace amounts of MU IKBKAP can be seen in the untreated WT lanes.PCR fragment sizes of the two spliced products are shown on the left.(c) RT-PCR of the same RNA in panel (b) using T7 and a primer that spansthe 19-21 exon junction known to specifically amplify the MU band⁴,showing absence of MU transcript in the WT lane following kinetintreatment. The size of this fragment is shown on the left.

DETAILED DESCRIPTION OF THE INVENTION

Novel methods are provided for increasing the amount of mRNA spliced ina wild type fashion and/or wild type protein in cells or individualsusing one or more compounds that alters the splicing of mRNAtranscripts. In preferred embodiments, the ratio of wild-type tomisspliced mRNA or wild type to mutant protein in a cell or individualis increased by administration of the one or more compounds. Methods ofthe invention can be used to prevent, manage, or treat disordersassociated with missplicing. In preferred embodiments, the compound is acytokinin, preferably a 6-(substituted amino) purine cytokinin, morepreferably benzyladenine, most preferably kinetin. In other embodiments,the compound is one that increases production of properly spliced mRNAto a degree that is substantially similar to or greater than kinetin.

Disorders Treated with Methods of the Invention

The invention further relates to methods for altering the splicing ofIKBKAP by contacting cells with a cytokinin, (preferably benzyladenineand more preferably kinetin). In particular, the invention relates toenhancing correct mRNA splicing in order to increase cellular levels ofnormal or wild type IKAP mRNA or protein encoded by a mutant IKBKAP genein lymphoblast, fibroblast and neuronal cells. In specific embodiments,the invention relates to the use of kinetin for increasing the inclusionof exon 20 from the IKBKAP gene in spliced mRNA transcripts. In morespecific embodiments, kinetin provides a treatment for individuals withFD by increasing the level of normal IKAP mRNA and protein.

The methods of the invention can be used to prevent, manage, or treatother disorders, in addition to FD, characterized by missplicing (seeTable 1 for non-FD disorders). The missplicing in the other disordersmay result from a mutation in i) the misspliced transcript, or ii) acomponent of the splicing machinery responsible for processing themisspliced transcript.

In a specific embodiment, the non-FD disorder prevented, managed, ortreated by the methods of the invention is NF1 caused by missplicing.NF1 gene splicing can be altered (e.g., to increase levels of wild typesplicing) by contacting cells with a cytokinin, (preferablybenzyladenine and more preferably kinetin).

TABLE 1 Misspliced mRNA Disorder transcript Mutant geneNeurofibromatosis 1 (NF1) NF1 NF1 Neurofibromatosis 2 (NF2) NF2 NF2Familial isolated growth growth hormone growth hormone hormonedeficiency type (GH-1) (GH-1) II (IGHD II) Frasier syndrome Wilms tumorWilms tumor suppressor suppressor gene (WT1) gene (WT1) Frontotemporaldemetia and tau (MAPT) tau (MAPT) Parkinsonism lined to Chromosome 17(FTDP-17) Atypical cystic fibrosis cystic fibrosis cystic fibrosistransmembrane transmembrane conductance conductance regulator (CFTR)regulator (CFTR) Menkes Disease (MD) ATP7A ATP7A Occipital Horn SyndromeATP7A ATP7A Myotonic dystrophy DM protein kinase type 1 (DM1) (DMPK)Myotonic dystrophy ZNF9 type 2 (DM2) Retinitis pigmentosa (RP) opsinPRPF31, HRRP3, or PRPC8 Spinal muscular Survivor of atrophy (SMA) MotorNeuron gene 2 (SMN2)

This invention further provides methods for treating individuals at riskfor developing a disorder associated with a misspliced mRNA transcript.In a preferred embodiments, the individual is at risk for developingfamilial dysautonomia or NF-1. Persons at risk for developing familialdysautonomia or NF1 may be identified by genetic screening for thepresence of a mutation associated with familial dysautonomia (see e.g.,International Patent Application No. PCT/US02/00473, filed Jan. 7, 2002,entitled: GENE FOR IDENTIFYING INDIVIDUALS WITH FAMILIAL DYSAUTONOMIAand which is incorporated herein by reference in its entirety) or NF-1caused by missplicing (see e.g., U.S. Pat. Nos. 5,227,292, 5,605,799,5,859,195, and 6,238,861). Accordingly, the compositions for use withthe methods of this invention may be administered to an individual atvarious times during the course of the disease and during differentdegrees of expression of clinical symptoms. In a preferred embodiment,this invention provides methods and compositions for treatingindividuals with and at risk for developing the degenerative symptomsassociated with familial dysautonomia, such as neuronal degeneration.

Compounds for use in the Methods of the Invention

Compositions administered for the treatment of a disorder associatedwith a misspliced mRNA transcript can comprise one or more compoundsthat increase the amount of mRNA transcript spliced in a wild typemanner and/or alter the ratio of wild type to misspliced mRNAtranscripts. In preferred embodiments, at least one of the compounds inthe composition is a cytokinin. In more preferred embodiment, thecytokinins are 6-(substituted amino)purine cytokinins. 6-(substitutedamino)purine cytokinins include, but are not limited to, benzyladenine,kinetin, and 6-amino analogs thereof of Formula I:

in which R₁ is furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons,branched alkyl of 4, 5, or 6 carbons, (cyclohexyl) methyl,3,3-dimethylallyl, and 3-hydroxymethyl-3-methylallyl. Among the6-(substituted amino)purine cytokinins that are intended to be used,singly or in combination, as a compound in the methods herein arekinetin, benzyladenine, isopentenyl adenine,(6-(3-hydroxymethyl-3-methylallyl)-aminopurine),6-(3,3-dimethylallyl)aminopurine, 6-(benzyl)aminopurine,6-(phenyl)aminopurine, 6-(n-alkyl)aminopurine, in which the n-alkylgroup has 4, 5 or 6 carbons, and 6-(cyclohexyl)methylaminopurine. Mostpreferred is kinetin (6-(furfuryl)aminopurine). Other such6-(substituted amine) purine cytokinins may be tested for the ability toimprove proper mRNA splicing in cells in vitro.

In other preferred embodiments, the one or more cytokinins areadministered to a subject in need thereof in combination with at leastone other compound that provides a therapeutic effect. Examples of suchother compounds include, but are not limited to, antioxidants (such as(−)-epigallocatechin gallate) and tocotrienols (such as α-tocotrienol,β-tocotrienol, γ-tocotrienol, and δ-tocotrienol). Such other compoundsto be administered in combination with the one or more cytokinins may ormay not be part of the same composition that comprises the one or morecytokinins.

The term “in combination” is not limited to the administration of thecompounds at exactly the same time, but rather it is meant that thecompounds are administered to a subject in a sequence and within a timeinterval such that they can act together to provide an increased benefitthan if they were administered otherwise. For example, each compound maybe administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect. Each compound can be administeredseparately, in any appropriate form and by any suitable route.

In various embodiments, the compounds are administered less than 1 hourapart, at about 1 hour apart, at about 1 hour to about 2 hours apart, atabout 2 hours to about 3 hours apart, at about 3 hours to about 4 hoursapart, at about 4 hours to about 5 hours apart, at about 5 hours toabout 6 hours apart, at about 6 hours to about 7 hours apart, at about 7hours to about 8 hours apart, at about 8 hours to about 9 hours apart,at about 9 hours to about 10 hours apart, at about 10 hours to about 11hours apart, at about 11 hours to about 12 hours apart, no more than 24hours apart or no more than 48 hours apart. In preferred embodiments,two or more compounds are administered within the same patient visit.

Those compounds that improve proper mRNA splicing either by increasingthe amount of wild type mRNA transcript and/or by increasing the ratioof wild-type to mutant mRNA or protein may be formulated aspharmaceuticals for administration to individuals or added to tissueculture medium at effective concentrations. Such concentrations arepreferably about 0.1 ppm to about 500 ppm, preferably 10 ppm to about100 ppm, in tissue culture medium and about 10 ppm to about 5000 ppm,preferably about 100 to about 1000 ppm, in pharmaceutical compositions.The precise concentrations, particularly for in vivo use, may bedetermined empirically and may be higher, particularly for in vivo use,depending upon the ability of the carrier or vehicle to deliver thecompound or compounds to the treated cells or tissue and the manner inwhich compositions is contacted with the treated cells or tissue.

Identification of Compounds for use in Methods of the Invention

The invention provides methods of screening for compounds that can altersplicing of a misspliced mRNA transcript, especially a mutant mRNAtranscript that is misspliced. In a specific embodiment, the mutant mRNAtranscript is a IKBKAP mRNA transcript. In a more specific embodiment,the a mutant IKBKAP mRNA transcript is present in a subject with FD. Inan even more specific embodiment, the mutant IKBKAP mRNA transcript hasthe IVS20 ^(+6T→C) mutation. In another specific embodiment, the mutantmRNA transcript is a NF1 mRNA transcript. In a more specific embodiment,the a mutant NF1 mRNA transcript is present in a subject with NF1.

Although not intending to be bound by a particular mechanism of action,a compound for use in the methods of the invention can alter thesplicing of a misspliced mRNA by i) increasing wild type splicing ofmutant mRNA transcripts, ii) decreasing mutant splicing of mutant mRNAtranscripts, iii) increasing the amount of wild type splicing of wildtype transcripts by mutant splicing machinery, and/or iv) decreasing theamount of mutant splicing of wild type mRNA transcripts by mutantsplicing machinery. In other embodiments, compounds for use in themethods of the invention can alter the amount of mutant protein producedfrom either a i) mutant mRNA transcript or ii) wild type mRNA transcriptspliced by mutant splicing machinery. Although not intending to be boundby a particular mechanism of action, a compound for use in the methodsof the invention can alter the amount of mutant protein produced from amisspliced mRNA transcript by i) increasing the translation of mRNAtranscripts spliced in a manner consistent with wild type transcripts,ii) decreasing the translation of mRNA transcripts spliced in a mannerinconsistent with wild type transcripts.

The methods of screening generally involve incubating a candidatecompound with animals or cells that express a misspliced mRNA transcriptand then assaying for an alteration in the splicing of the missplicedmRNA transcript thereby identifying a compound for use in the methods ofthe invention. The DNA comprising the gene which is misspliced may beendogenous or it may be heterologous, e.g., contained on a vector whichhas been inserted into the cell used in the assay such as bytransfection or transduction or contained in a transgene which has beenused to make a transgeneic. In embodiments where the DNA comprising thegene which is misspliced is heterologous, fragments of the full lengthgene may be used comprising at least the portion of the gene that ismisspliced. In more specific embodiments, the fragment comprises exon 20of IKBKAP.

In some embodiments, the amount of wild type spliced mRNA, mutantspliced mRNA, and/or both is determined. Any method known in the art canbe used to assay for levels of mRNA transcripts, including, but notlimited to, those assays to detect i) mRNA levels (e.g., by northernblots, RT-PCR, Q-PCR, etc.) or ii) protein levels (e.g., ELISA, westernblots, etc.). In specific embodiments, an increase in the ratio of wildtype mRNA transcripts to misspliced mRNA transcripts indicates that thecompound decreases missplicing. In another specific embodiment, anincrease in the ratio of wild type spliced mRNA transcripts to a controlgene transcripts indicates that the compound decreases missplicing. Inanother specific embodiment, a decrease in the ratio of misspliced mRNAtranscripts to a control gene transcripts indicates that the compounddecreases missplicing. As used herein, the term “control gene” refers toa gene whose splicing or expression are not altered by any mutation thatthe animal or cell used in the assay may have or by contact of thecandidate compound. Control genes may be endogenous (e.g. actin, etc.)or heterologous (e.g., a reporter gene such as luciferase, GFP, CAT, orβ-galactosidase,ect.).

In other embodiments, the fragment of the misspliced gene comprising theportion that is misspliced may be part of a fusion protein with areporter gene (e.g., luciferase, GFP, CAT, or β-galactosidase). Such afusion protein will allow a signal from the reporter gene if the portionof the misspliced gene has been removed due to splicing. No signal or areduced signal will be present from the reporter gene if the portion ofthe misspliced gene has not been removed due to splicing. If a candidatecompound decreases missplicing, then the exon that is normally excludeddue to missplicing will be included in the fusion protein and thusdecrease the signal from the reporter gene. In a specific embodiment,the reporter gene comprises exon 20 of IKBKAP. In another specificembodiment, the reporter gene comprises exon 36 of NF1.

In some embodiments, expression of a misspliced mRNA transcript confersa phenotype to the animal or cell expressing the transcript that can beassayed (e.g., altered growth rate, longevity, behavior, etc.).Candidate compounds that can be used in the methods of the inventionwill cause a change in at least one of the misspliced mRNAtranscript-associated phenotypes. In a preferred embodiment, the changein the misspliced mRNA transcript-associated phenotype is such that itapproximates (or is substantially similar) to that of an organism orcell expressing a corresponding wild type mRNA transcript. In otherembodiments, candidate compounds are assayed for their ability to alterthe splicing of misspliced mRNA transcripts and/or alter misspliced mRNAtranscript-associated phenotypes in a manner that is substantiallysimilar to or better than compounds known to alter the splicing ofmisspliced mRNA transcripts in a therapeutically desirable way (e.g.,cytokinins including kinetin). As used herein “substantially similar to”refers to a ratio of wild type to misspliced mRNAs and/or a missplicedmRNA transcript-associated phenotype that is more similar to that of acell or organism i) expressing a wild type counterpart of the missplicedmRNA transcript or ii) expressing the misspliced mRNA transcript treatedwith a cytokinin (especially kinetin) than a cell or organism expressingthe misspliced mRNA transcript and not treated with a cytokinin. Anyanimal model known in the art can be used to assay candidate compoundsincluding, but not limited to, those described in Costa et al., 2002,Nature 415: 526-30 and Costa et al., 2001, Nat Genet. 27:399-405.

The screening methods of the invention also encompass the use ofbiochemical assays (e.g., in vitro transcription and/or translationassays) to identify compounds. Candidate compounds found to alter thesplicing of misspliced mRNA in biochemical assays can then be assayed inanimal or cell-based assays to determine any phenotype-alteringproperties.

The screening methods of the invention may be adapted for use in highthroughput screen for compositions that can be effective for thetreatment of disorders associated with missplicing of mRNAtranscript(s), especially familial dysautonomia and NF1.

As used herein, the term “compound” refers to a molecule that has adesired biological effect. Compounds include, but are not limited to,proteinaceous molecules, including, but not limited to, peptide,polypeptide, protein, post-translationally modified protein, antibodiesetc.; or a large molecule, including, but not limited to, inorganic ororganic compounds; or a small molecule (less than 500 daltons),including, but not limited to, inorganic or organic compounds; or anucleic acid molecule, including, but not limited to, double-strandedDNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, ortriple helix nucleic acid molecules. Compounds can be natural productsderived from any known organism (including, but not limited to, animals,plants, bacteria, fungi, protista, or viruses) or from a library ofsynthetic molecules.

In one embodiment, a compound that decreases the amount of a missplicedmRNA transcript is identified by:

-   -   a) contacting a cell or organism with a compound, wherein said        cell or organism expresses said misspliced mRNA transcript; and    -   b) determining the ratio of wild type to misspliced mRNA        transcripts in said contacted cell or organism,        wherein an increase in the ratio of wild type to misspliced mRNA        transcripts of said contacted cell or organism as compared to        the ratio of wild type to misspliced mRNA transcripts of a cell        or organism expressing said misspliced mRNA transcript not        contacted with the compound (i.e., a control cell or organism)        indicates that the compound decreases the amount of said        misspliced mRNA transcript.

In another embodiment, a compound that decreases the amount of amisspliced mRNA transcript is identified by:

-   -   a) contacting a cell or organism with a compound, wherein said        cell or organism expresses said misspliced mRNA transcript; and    -   b) determining the ratio of wild type to misspliced mRNA        transcripts in said contacted cell or organism,        wherein the ratio of wild type to misspliced mRNA transcripts of        said contacted cell or organism is substantially similar to the        ratio of wild type to misspliced mRNA transcripts of a cell or        organism expressing said misspliced mRNA transcript contacted        with kinetin indicates that the compound decreases the amount of        misspliced mRNA transcript.

In another embodiment, a compound that alters the amount of a missplicedmRNA transcript is identified by:

-   -   a) contacting a cell or organism with a compound, wherein said        cell or organism exhibits at least one phenotype that is altered        as a result of its expression of said misspliced mRNA transcript        when compared to a wild type cell or organism; and    -   b) determining the phenotype of said contacted cell or organism,        wherein a difference in the phenotype of said contacted cell or        organism as compared to the phenotype of a cell or organism        expressing said misspliced mRNA transcript not contacted with        the compound (i.e., a control cell or organism) indicates that        the compound alters the amount of said misspliced mRNA        transcript.

In another embodiment, a compound that decreases the amount of amisspliced mRNA transcript is identified by:

-   -   a) contacting a cell or organism with a compound, wherein said        cell or organism exhibits at least one phenotype that is altered        as a result of its expression of said misspliced mRNA transcript        when compared to a wild type cell or organism; and    -   b) determining the phenotype of said contacted cell or organism,        wherein the phenotype of said contacted cell or organism is        substantially similar to the phenotype of a cell or organism        expressing said misspliced mRNA transcript contacted with        kinetin indicates that the compound decreases the amount of        misspliced mRNA transcript.

Treatment of Disorders Using Methods of the Invention

Disorders associated with misspliced mRNA can be prevented, managed, ortreated by the methods of the invention. Individuals suffering from orlikely to suffer from such a disorder are administered compositionscomprising compounds that have a desired therapeutic effect (e.g.,increasing the amount mRNA spliced in the wild type fashion). Disordersthat can be treated by the methods of the invention include, but are notlimited to, FD, NF1 cased by missplicing, and those disorders listed inTable 1. Compositions for the treatment of such disorders comprise oneor more cytokinins at concentrations effective to produce a therapeuticeffect. In preferred embodiments, at least one of the one or morecytokinins is a 6-(substituted amino)purine cytokinins. In morepreferred embodiments, the 6-(substituted amino)purine cytokinin iskinetin or benzyladenine. Compositions administered to individuals inneed thereof can further comprise other compounds that have a desiredtherapeutic effect including, but not limited to tocotrienols (e.g.δ-tocotrienol) and/or antioxidents (e.g., (−)-epigallocatechin gallate)in order to ameliorate or correct the adverse effects in an individualresulting from improperly spliced mRNA. In other embodiments,compositions for the treatment of disorders associated with missplicingcomprise a therapeutically effect amount of a compound that increasesproduction of properly spliced mRNA to a degree that is substantiallysimilar to or greater than kinetin.

Compositions comprising compounds that have a desired therapeutic effectcan also be administered to cells in vitro. Such cells in tissue cultureprovide a useful means for assessing the effectiveness of candidatecompounds for increasing the ratio of wild type to mutant protein. Suchcells may be either from tissue explants, or immortalized cell cultures.Examples of specific cell types include but are not limited to,lymphoblast, fibroblast and neuronal. The cells in culture can bebathed, suspended or grown in a culture medium used for mammalian cells.The medium contains an effective concentration of the compositioncomprising the compounds with therapeutic effect (e.g., one or more6-(substituted amino)purine cytokinins selected from the groupconsisting of kinetin, benzyladenine, isopentenyl adenine,(6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),6-(3,3-dimethylallyl)aminopyrine, 6-(benzyl)aminopyrine,6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkylgroup has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and thosecompounds of Formula I,

in which R₁ is furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons,branched alkyl of 4, 5, or 6 carbons, (cyclohexyl) methyl,3,3-dimethylallyl, and 3-hydroxymethyl-3-methylallyl). Preferredcompounds, include, but are not limited to, kinetin and benzyladenine,most preferred is kinetin. A preferred concentration of the compounds offormula I in the medium is about 0.1 ppm to about 500 ppm, morepreferably about 0.1 to 100 ppm, or a concentration of equivalentactivity to a kinetin concentration of between about 10⁻⁶ M (1 μM orabout 5 ppm) to about 5×10⁻⁴ M (50 μM or about 250 ppm). For the mostpreferred cytokinin, kinetin, the more preferred concentration range isabout 25 μM to about 250 μM or about 5 ppm to about 50 ppm in theculture medium. It is understood that the precise concentration for each6-(substituted amino) purine cytokinin or mixture thereof may beempirically determined by testing a range of concentration and selectingthose in which the ratio of wild-type to misspliced mRNA or protein isincreased.

Additionally, such treated cells may be administered to an individual asex vivo therapy either in addition to or instead of administration of acomposition comprising the compounds with a desired therapeutic effect.

Administration

The compositions may be formulated in numerous forms, depending on thevarious factors specific for each patient (e.g., the severity and typeof disorder, age, body weight, response, and the past medical history ofthe patient), the one or more compounds in the composition, the form ofthe compounds (e.g., in liquid, semi-liquid or solid form), and/or theroute of administration (e.g., oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, or rectal means). Pharmaceutical carriers,vehicles, exipients, or diluents may be included in the compositions ofthe invention including, but not limited to, water, saline solutions,buffered saline solutions, oils (e.g., petroleum, animal, vegetable orsynthetic oils), starch, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene, glycol, ethanol,dextrose and the like. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be provided as a liquid suspension or solution orcompressed into tablets or enclosed in gelatin capsules. For the purposeof oral therapeutic administration, the active compound or compounds canbe incorporated with excipients and used in the form of solutions orsuspensions, tablets, capsules or troches. The tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder, such as microcrystallinecellulose, gum tragacanth and gelatin; an excipient such as starch andlactose, a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a glidant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, and fruitflavoring. Further details on techniques for formulation andadministration are provided in the latest edition of Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors. The active materials can also be mixed with otheractive materials which do not impair the desired action, or withmaterials that supplement the desired action.

Solutions or suspensions used for oral administration can include any ofthe following components: a sterile diluent, such as water forinjection, saline solution, fixed oil, polyethylene glycol, glycerine,propylene glycol or other synthetic solvent; antimicrobial agents, suchas benzyl alcohol and methyl parabens; antioxidants, such as ascorbicacid and sodium bisulfite; chelating agents, such asethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Liquid preparations can be enclosed inampules, disposable syringes or multiple dose vials made of glass,plastic or other suitable material. Suitable carriers may includephysiological saline or phosphate buffered saline (PBS), and thesuspensions and solutions may contain thickening and solubilizingagents, such as glucose, polyethylene glycol, and polypropylene glycoland mixtures thereof. Liposomal suspensions, including tissue-targetedliposomes, may also be suitable as pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. In addition,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyloleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants or permeation agentsthat are appropriate to the particular barrier to be permeated are usedin the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Saltstend to be more soluble in aqueous solvents, or other protonic solvents,than are the corresponding free base forms.

Dosages

The compounds for use in the methods of the invention are present incompositions in an amount sufficient to have a therapeutic effect on thetreated individual without serious toxic effects. The determination ofan effective concentration or dose is well within the capability ofthose skilled in the art. The effective concentration or dose may bedetermined empirically by testing the compounds in individuals who wouldbenefit from treatment, or using in vitro and in vivo systems, includingtissue culture (e.g., using lymphoblast, fibroblast, or neuronal cells)or suitable animal models.

A therapeutically effective dose refers to that amount of a compound(e.g., cytokinin such as kinetin or other 6-(substituted amino) purinecytokinin) which prevents, ameliorates, reduces, or eliminates thesymptoms of a disorder associated with a misspliced mRNA. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe ratio, ED50/LD50. Pharmaceutical compositions, which exhibit largetherapeutic indices, are preferred. The data obtained from cell cultureassays and animal studies are used in determining a range of dosages forhuman use. Preferred dosage contained in a pharmaceutical composition iswithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

The exact dosage will be determined by the practitioner, who willconsider the factors related to the individual requiring treatment.Dosage and administration are adjusted to provide sufficient levels ofthe active compound or to maintain the desired effect of the activecompound. Factors, which may be taken into account, include the severityof the individual's disease state, general health of the patient, age,weight, and gender of the patient, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Guidance as to particular dosages andmethods of delivery is provided in the literature and is generallyavailable to practitioners in the art.

The concentration of active compound in the compositions will depend onabsorption, inactivation, excretion rates of the active compound, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art. Typically a therapeutically effectivedosage should deliver a concentration of at least about 10 ppm up toabout 5000 ppm, preferably 50 ppm to about 1000 ppm, of the activecompound to the treated individual. The active ingredient may beadministered at once, or may be divided into a number of smaller dosesto be administered at intervals of time. It is understood that theprecise dosage of treatment is a function of individual being treatedand may be determined empirically using known testing protocols or byextrapolation from in vivo or in vitro test data. It is to be furtherunderstood that for any particular individual, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the medical personal administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the described compositions.

In certain embodiments, compositions are effective at concentrations ofthe 6-(substituted amino)purine cytokinin typically in the range ofbetween about 0.1 ppm to about 100 ppm. When kinetin is used, thepreferred concentration range is between about 10⁻⁶ M and 5×10⁻⁴ M inthe blood. At these concentrations, cytokinins, apparently have no orminimal toxic effect on mammalian cells in tissue culture. Inembodiments where δ-tocotrienol is administered in combination with theone or more cytokinins, the preferred concentration range is between0.25 μg/ml and 50 μg/ml in the blood. In embodiments where(−)-epigallocatechin gallate is administered in combination with the oneor more cytokinins, the preferred concentration range is between 5 μg/mland 60 μg/ml in the blood.

EXAMPLES Materials and Methods

Patient cell lines. Patient lymphoblast cell lines previouslyestablished by EBV transformation were utilized. The cells have beenpreviously described. ^(2,14). Institutional Review Board approval hadbeen obtained for the establishment and use of these lines through NewYork University Medical Center, Massachusetts General Hospital, andHarvard Medical School. Two fibroblast cell lines were also used.GUS12829 was established and has been previously described. GM02343 waspurchased from the Coriell Cell Repository.

Drug Screen. The panel of 1040 independent compounds used wasspecifically designed for the NINDS sponsored Neurodegeneration DrugScreening Consortium (MicroSource Discovery Systems). A single FDlymphoblast cell line was cultured in 24 well format and treated with 10μM compound dissolved in DMSO for 72 hours. RNA was extracted usingTri-Reagent™(Molecular Research Center) and reverse transcription andIKBKAP PCR was performed as previously described⁴.

Cell Culture and Treatment. FD lymphoblast lines were grown in RPMI-1640and primary fibroblast lines in Dulbecco's modified Eagle's media (DMEM)with Earle's balanced salts. Both media were supplemented with 2 mML-glutamine and 1% penicillin/streptomycin and either 10% (lymphoblast)or 20% (fibroblast) fetal bovine serum (Invitrogen). Kinetin wasobtained from both MicroSource Discovery Systems and Sigma and dissolvedat 10 mM in either DMSO or water. FD cells were cultured in the presenceof kinetin for 72 hours except where noted. Benzyladenine, zeatin, and2iP were obtained from Sigma and dissolved in DMSO at 10 mM. Thesecompounds were tested at concentrations of 1 μM, 10 μM, 50 μM, and 100μM. Cycloheximide was obtained from Sigma and dissolved at 10 mg/ml inDMSO. Cells were treated with 100 μM kinetin for eight hours, then 50μg/ml cycloheximide was added to the cultures for four hours¹⁶.

Determination of IKBKAP transcript ratios. The ratio of WT:MU IKBKAPtranscripts was determined following amplification by fractionation on1.5% agarose gels stained with ethidium bromide⁴. Each gel band wasassessed using an Alpha 2000™ Image Analyzer and software coupled withautomatic background subtraction (Alpha Innotech Corporation). WT:MUratios were obtained using the integrated density value (IDV) for eachband⁴. Real-time quantitative PCR (QPCR) was performed as describedusing primers specific to WT IKBKAP, MU IKBKAP, and 18S ribosomal RNA⁴.In order to assess total IKBKAP expression the following primers wereused: EX3F-5′-TCAGGACTTGCTGGATCAGGA (SEQ ID NO:1) and EX4R-5′-CCACTGGCTACACTCCCTTCT (SEQ ID NO:2) located in IKBKAP exons 3 and 4and an exon 3 TaqMan probe: TCTGGAGACGTCATACTCTGCAGTCTCAGC (SEQ IDNO:3). For the Myosin VA assay, primers were designed that flank thealternatively spliced exon 31 in order to assess transcript levels ofthe two isoforms. Primer sequences were as follows: MYO-F: GAA TAC AATGAC AGA TTC CAC (SEQ ID NO:4); MYO-R: CAG GCT GGC CTC AAT TGC (SEQ IDNO:5). Following reverse-transcription and PCR, products were separatedon a 1.5% agarose gel and IDV ratios determined as described above.

Western Blot Analysis. Patient lymphoblast cell lines were treated withincreasing concentrations of kinetin for 72 hours. Extracted protein wasrun on a 6% polyacrylamide gel. The samples were transferred tonitrocellulose and stained with Ponceau-S to visualize protein loading.IKAP was detected using a monoclonal antibody raised against amino acids796-1008 of IKAP (BD Bioscience) that detects the 150 kD full-lengthIKAP protein. The same blot was probed with an antibody toinsulin-degrading enzyme (IDE) as a protein loading control. Bands werequantitated using an Alpha 2000™ Image Analyzer.

In vivo IKBKAP minigene splicing assay. IKBKAP genomic DNA was amplifiedfrom an unaffected and an FD individual using primers in exon 19 and 21:EX19F -5′-CATTACAGGCCGGCCTGAG (SEQ ID NO:6) and EX21R-5′-CAGCTTAGAAAGTTACCTTAG (SEQ ID NO:7). The amplified products werecloned into pcDNA3.1/V5-His Topo (Invitrogen) and sequenced forverification. HEK293 cells were plated and kinetin was added to thetissue culture media 4 hours later. Minigene constructs were transientlytransfected 12 hours later using Genejuice (Novagen) as directed by themanufacturer. After 48 hours RNA was isolated using the RNAeasy kit(Qiagen) and reverse transcribed using Superscript™ II reversetranscriptase (Invitrogen) as described⁴. PCR was performed using vectorspecific primers: T7 (TAATACGACTCACTATAGG) (SEQ ID NO:8) and BGHR(TAGAAGGCACAGTCGAGG) (SEQ ID NO:9), which amplify both WT and MUtranscripts, or the MU specific primer EX19/21 F (CAAAGCTTGTATTACAGACT)(SEQ ID NO:10) and BGH-R to specifically amplify only MU transcripts.PCR was performed as follows: 26 cycles of [94° C., 30 s; 56° C., 30 s;72° C., 30 s] and products resolved on a 1.5% agarose gel and visualizedby ethidium bromide staining.

Example 1 IKBKAP mRNA Ratio Analysis

An assay that provides stable and consistent measurement of the ratio ofWT:MU FD IKBKAP mRNA splice products in lymphoblast cell lines wasdeveloped⁴. This assay was employed as part of the NeurodegenerationDrug Screening Consortium⁷ to identify compounds that increase therelative production of WT mRNA. A single, standard FD lymphoblast cellline was assayed after 72 hours in the presence of 10 μM test drug,using compounds dissolved in DMSO. A panel of 1040 bioactive compounds(NINDS Custom Collection, MicroSource Discovery Systems) were screened,most of which have been approved for use by the Food and DrugAdministration (FDA). Prior to initiating the screen, it was confirmedthat DMSO had no effect on IKBKAP splicing and was not toxic to thecells at the test concentration of 0.1%. One compound, kinetin(6-furfurylaminopurine) (FIG. 1 b), dramatically enhanced correctsplicing in FD cells at the test concentration of 10 μM (FIG. 1 c).

Concentrations of kinetin up to 100 μM were tested and significantenhancement of WT IKBKAP production with increasing concentration in twoindependent FD lymphoblast lines was observed (FIG. 2 a). The increasein WT:MU mRNA was mirrored by an increase in production of IKAP protein(FIG. 2 b), confirming the increased production of functional WT mRNA.To avoid DMSO toxicity at higher concentrations, kinetin was purchasedfrom an independent source (Sigma) and dissolved in water. Real-timequantitative PCR analysis showed that the WT:MU IKBKAP mRNA ratioincreased steadily from approximately 1:1 in untreated cells to 12:1 incells treated with 400 μM kinetin, the maximum dose tested due to theappearance of some cellular toxicity (FIG. 2 c). By contrast, totalIKBKAP mRNA was relatively unaffected, with a slight increase (˜1.5fold) being detected only at doses higher than 50 μM (data not shown).In an FD lymphoblast assay, neither zeatin nor 2iP (dissolved in DMSO at10 mM) had any effect on WT:MU IKBKAP ratio. Benzyladenine did enhanceinclusion of exon 20 in the IKBKAP transcripts approximately 2-fold by100 μM, a less dramatic effect than kinetin. The fact that only kinetinand benzyladenine alter IKBKAP splicing suggests that the nature of theadenine N⁶-linked modification plays a role in this function.

To define how quickly kinetin has its effect on IKBKAP splicing, atime-course at 50 μM kinetin was performed (FIG. 2 d). The increase inthe WT:MU ratio was seen at one hour, was maximal by 8 hours, and wasmaintained for at least 72 hours in culture without kinetinreplenishment. The consistency of the effect was tested by treatinglymphoblast lines from nine FD patients with 50 μM kinetin for 24 hours.All nine lines showed an increase in WT IKBKAP mRNA with WT:MU ratiosranging from 1.3-1.9 in untreated cells to 5.6-8.6 in kinetin treatedcells. Kinetin was also effective in FD fibroblast lines which exhibiteda dose-dependent increase in WT:MU IKBKAP ratio similar to that seen inlymphoblasts.

Example 2 MYO5A mRNA Ratio Analysis

To explore whether the effect of kinetin was specific to IKBKAP exon 20or generally increased inclusion of alternatively spliced exons,splicing of the MYO5A gene was assayed (FIG. 3 a). MYO5A expressesmultiple isoforms in fibroblasts and lymphoblasts, one of which isgenerated by skipping of exon 31¹⁰. Primers flanking exon 31 weredesigned. The ratio of the two MYO5A isoforms in nine FD lymphoblastcell lines following treatment with 50 μM kinetin for 72 hours wasexamined. No significant difference was observed in the ratio of the twoisoforms between the treated and untreated samples, with averagetranscript ratios of 2.33 and 2.46, respectively (FIG. 3 b).

Example 3 IKBKAP Nonsense Mediated Decay Analysis

The IVS20^(+6T→C) mutation in IKBKAP leads to a frameshift and apremature termination codon in exon 21, which is expected to target themutant transcript for decay via the nonsense-mediated mRNA decay (NMD)pathway¹⁵. To exclude the possibility that the observed changes in theWT:MU transcript ratio in the presence of kinetin were due to increasedNMD of the mutant transcript rather than direct action of kinetin onIKBKAP splicing, the WT:MU transcript ratio was assayed in the absenceof NMD. FD cells were exposed to cycloheximide, a translation inhibitor,to inhibit NMD of the mutant transcript¹⁶. FD cells treated withcyclohexamide alone increased mutant transcript levels therebydecreasing the WT:MU transcript ratio as expected (FIG. 4). However, FDcells treated kinetin in the presence of cycloheximide significantlyaltered the WT:MU transcript ratio in a manner similar to that ofkinetin treatment alone (FIG. 4). Thus, the observed decrease in theWT:MU transcript ratio in kinetin-treated FD cells is not dependent onNMD-mediated destruction of the mutant transcript, but rather is due tokinetin's action on IKBKAP splicing.

Example 4 IKBKAP Minigene Analysis

For studies aimed at understanding the mechanism of action of kinetin onIKBKAP splicing, both FD and WT minigene constructs containing thegenomic sequence spanning exon 19 to 21 were created (FIG. 5 a). Theseconstructs were transfected into HEK293 cells and RNA was amplifiedusing vector-specific primers to avoid amplification of the endogenousIKBKAP message. Evaluation of the transcripts produced from the WTconstruct showed that although the WT transcript is the predominantproduct, trace levels of exon 20 skipping do occur even in the absenceof the FD mutation. Introduction of the FD mutation into the constructincreased exon 20 skipping as predicted. Interestingly, treatment withkinetin enhanced inclusion of exon 20 not only in the FD construct, butalso in the WT construct (FIG. 5B). In fact, utilization of aMU-specific PCR assay⁴ revealed that exon 20 skipping is completelycorrected in transcripts generated from the WT construct by kinetintreatment (FIG. 5 c). These findings indicated that the ability ofkinetin to enhance splicing efficiency is not dependent on either thepresence of the FD mutation or the wider regulation of IKBKAPtranscription or IKAP protein production, and is likely due to specificsequence elements in the region of exon 20 that function to regulatesplicing of this particular exon.

Example 5 SMN1 and MYO5A Analysis

Comparison of SMN1 and SMN2, closely related genes involved in spinalmuscular atrophy (SMA), has shown that a single DNA change in the codingsequence results in skipping of exon 7 in SMN2^(11,12). It has beenreported that aclarubicin treatment can promote the inclusion of exon 7in the SMN2 transcript¹³. Aclarubicin was tested on FD cells as part ofour original screen and had no effect on IKBKAP splicing. Similarly,kinetin has no effect on skipping of exon 7 in SMN2 (Jianhua Zhou).Further, neither of these drugs promotes inclusion of alternativelyspliced exons in MYO5A¹³. Taken together, these studies clearly indicatethat there are multiple distinct mechanisms that contribute to exonchoice during pre-mRNA splicing in both normal and disease situations.The distinctive tissue-specific splicing pattern seen in FD and theknown importance of tissue-specific alternative splicing in generatingprotein diversity add yet another level of complexity to this importantregulatory process.

Example 6 Clinical Assessment for Treatment of Familial Dysautonomia

Kinetin Drug Absorption Study. A drug absorption study is performed onapproximately 50 individuals. The individuals are given a dose ofkinetin Serum levels are obtained at various time points, 0 15 min, 30min, 60 min and 120 min, to determine clearance.

The assessment is an unblinded and longitudinal study where each patientis his or her own control. However, a blinded assessment with a placebocontrol could also be performed. A daily dose of kinetin is given forindividuals diagnosed with FD whose weight is greater than 25 kg.Dosages are calculated based on drug absorption assessment. An initialdose is calculated according to patient's weight and is administeredorally, via a gastrointestinal tube or sublingually, preferably orally.Blood samples will be taken at baseline (0 min), 15 min, 30 min and thenone hour and 2 hours post administration to assess blood level.

During the study, individuals are assessed at various time periods,including an initial visit, a visit at 1 month, 6 months, 12 months, 18months, 24 months and 36 months. Activities that occur at the variousvisits include functional assessment, determination of the wild-type tomutant ratio of mRNA IKAP or protein IKAP, sensory and gait tests,autonomic (CV and eye) tests and autonomic (EGG).

The functional assessment of the individuals is based on a score from 0(normal) to 15 (globally severely limited). The individuals oral intake,crisis frequency, cognitive ability, speech and gait are assessed.

In addition, blood tests are conducted to assess the mRNA IKAP ratio(wildtype:mutant) from isolated lymphoblasts. The ratio of wild type tomutant protein may also be assessed using methods well known in the artin combination with the methods described above. Alternatively oradditionally, other tissue types may be obtained by biopsy, includingbut not limited to a skin punch test, to assess the mRNA or protein IKAPwild type to mutant ratio found in fibroblasts.

Sensory assessments are also performed. The sensory assessments mayinclude the following: a histamine test (with photograph and measurementan appropriate time), a pain test (sharp vs. dull)*, a temperatureassessment (hot and cold thresholds using Thermotest by Nicolet)*,vibration thresholds (using biothesiometer)*, and deep tendon reflexes.(Items with * can only be done in patients over 6 years).

Autonomic assessments are also performed. The autonomic assessments mayinclude cardiovascular tests, including tilt test, heart ratevariability, sympathovagal balance, and autonomic perturbations. Morespecifically the tilt test includes blood pressure, mean blood pressureand heart rate while the individual is first supine and then erect for ashort and longer period of time. The heart rate variability test isperformed supine and erect and uses the Nicolet MMP program. Thesympathovagal balance test* is performed supine and erect and uses ANSARtechnology which provides SDNN and PN50 values (measures of sympatheticand parasympathetic tone, respectively). The autonomic perturbations*include deep breathing and valsalva, and tilt to assess sympathovagalresponsiveness (data attained via Atlas technology). Autonomicassessment also includes an ophthalmologic test—the Schirmer tear test.In addition, the autonomic assessment includes gastrointestinal testingcomprising a electrogastrogram with water load.

The extent of a response to treatment is based on a positive change inany objective measure of clinical symptom or biochemical marker duringtreatment. Such biochemical marker may include a change in protein or achange in mRNA ratio of wild type to mutant IKAP.

Clinical symptoms are assessed according to any method known in the artincluding Hilz et al., 1998, Journal Neurology, Neurosurgery, andPsychiatry.65:338-343; . Pearson and Pytel, 1978, J Neurol Sci 39:123-130; Axelrod, 1996, Autonomic and Sensory Disorders. In: Principlesand Practice of Medical Genetics, 3rd edition, Emory and Rimoin eds.Churchill Livingstone, Edinburgh. pp 397-411;. Axelrod, 2002, Clin AutonRes 12:2-14; Axelrod and Maayan, Familial Dysautonomia. In: Burg et al,eds, Gellis and Kagan's Current Pediatric Therapy, 17^(th) edition WBSaunders, Philadelphia, 2002, pp 437-441; Brown et al., 2003, ClinicalScience 104:163-9; Maayan et al., 1987, J. Auton Nerv. Syst. 21:51-8;Marthol et al., 2003, Eur. J. Clin. Invest. 33:912-8; Cuajungco et al.,2003, AM J Hum Genet. 72:749-58.

Absorption studies and symptom assessment are further conducted withkinetin in combination with δ-tocotrienol and/or (−)-epigallocatechingallate.

Example 7 NF1 Minigene Analysis

For studies aimed at understanding the mechanism of action of kinetin onNF1 splicing, both NF1 and WT minigene constructs containing the genomicsequence spanning exons 35 to 37 of the NF1 gene were created. NF1genomic DNA was amplified from an unaffected and an NF1 individual usingprimers at the 5′ end of exon 35 and the 3′ end of exon 37. The NF1individual had a C>T mutation at nucleotide residue position 6724 of theNF1 cDNA. The amplified products were cloned into pcDNA3.1/V5-His Topo(Invitrogen) and sequenced for verification. HEK293 cells were platedand kinetin was added to the tissue culture media 4 hours later.Minigene constructs were transiently transfected 12 hours later usingGenejuice (Novagen) as directed by the manufacturer. After 48 hours RNAwas isolated using the RNAeasy kit (Qiagen) and reverse transcribedusing Superscript™ II reverse transcriptase (Invitrogen) as described⁴.PCR was performed using vector specific primers: T7(TAATACGACTCACTATAGG) (SEQ ID NO:8) and BGHR (TAGAAGGCACAGTCGAGG) (SEQID NO:9), which amplify both WT and MU transcripts. PCR was performed asfollows: 30 cycles of [94° C., 30 s; 58° C., 30 s; 72° C., 30 s] andproducts resolved on a 1.5% agarose gel and visualized by ethidiumbromide staining.

When treated with kinetin, cells transfected with the NF1 minigene hadaltered splicing as compared to cells transfected with the NF1 minigenein the absence of kinetin. One of skill in the art could determine ifthe alteration in splicing resulted in more wild type and/or less mutantsplicing to occur using well known molecular biological techniques.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. The inventionnow being fully described, it will be apparent to one of ordinary skillin the art that many changes and modifications can be made theretowithout departing from the spirit or scope of the appended claims.

REFERENCES

-   1. Nissim-Rafinia, M. & Kerem, B. Splicing regulation as a potential    genetic modifier. Trends Genet 18, 123-7 (2002).-   2. Slaugenhaupt, S. A. et al. Tissue-specific expression of a    splicing mutation in the IKBKAP gene causes familial dysautonomia.    Am J Hum Genet 68, 598-605 (2001).-   3. Anderson, S. L. et al. Familial dysautonomia is caused by    mutations of the IMP gene. Am J Hum Genet 68, 753-8 (2001).-   4. Cuajungco, M. P. et al. Tissue-Specific Reduction in Splicing    Efficiency of IKBKAP Due to the Major Mutation Associated with    Familial Dysautonomia. Am J Hum Genet 72, 749-58 (2003).-   5. Maayan, C., Kaplan, E., Shachar, S., Peleg, O. & Godfrey, S.

Incidence of familial dysautonomia in Israel 1977-1981. Clin Genet 32,106-8 (1987).

-   6. Leyne, M., Mull, J., Gill, S. P., Cuajungco, M. P., Oddoux, C.,    Blumenfeld, A., Maayan, C., Gusella, J. F., Axelrod, F. B.,    Slaugenhaupt, S. A. Identification of the first non-Jewish mutation    in Familial Dysautonomia. Am J Med Genet In Press(2003).-   7. Heemskerk, J., Tobin, A. J. & Bain, L. J. Teaching old drugs new    tricks.

Meeting of the Neurodegeneration Drug Screening Consortium, 7-8 April2002, Washington, D.C., USA. Trends Neurosci 25, 494-6 (2002).

-   8. Rattan, S. I. & Clark, B. F. Kinetin delays the onset of ageing    characteristics in human fibroblasts. Biochem Biophys Res Commun    201, 665-72 (1994).-   9. Olsen, A., Siboska, G. E., Clark, B. F. & Rattan, S. I.    N(6)-Furfuryladenine, kinetin, protects against Fenton    reaction-mediated oxidative damage to DNA. Biochem Biophys Res    Commun 265, 499-502 (1999).-   10. Lambert, J., Naeyaert, J. M., Callens, T., De Paepe, A. &    Messiaen, L. Human myosin V gene produces different transcripts in a    cell type-specific manner. Biochem Biophys Res Commun 252, 329-33    (1998).-   11. Lorson, C. L., Hahnen, E., Androphy, E. J. & Wirth, B. A single    nucleotide in the SMN gene regulates splicing and is responsible for    spinal muscular atrophy. Proc Natl Acad Sci USA 96, 6307-11 (1999).-   12. Monani, U. R. et al. A single nucleotide difference that alters    splicing patterns distinguishes the SMA gene SMN1 from the copy gene    SMN2. Hum Mol Genet 8, 1177-83 (1999).-   13. Andreassi, C. et al. Aclarubicin treatment restores SMN levels    to cells derived from type I spinal muscular atrophy patients. Hum    Mol Genet 10, 2841-9 (2001).-   14. Blumenfeld, A. et al. Precise genetic mapping and haplotype    analysis of the familial dysautonomia gene on human chromosome 9q31.    Am J Hum Genet 64, 1110-8 (1999).-   15. Frischmeyer, P. A. and Dietz, H. C. (1999) Nonsense-mediated    mRNA decay in health and disease. Hum. Mol. Genet. 8, 1893-1900.-   Noensie, E. N. and Dietz, H. C. (2001) A strategy for disease gene    identification through nonsense-mediated mRNA decay inhibition. Nat.    Biotechnol. 19, 434-439.

1-45. (canceled)
 46. A method for treating neuronal degeneration in asubject in need thereof comprising administering to said subject acomposition that comprises an effective concentration, of one or more6-(substituted amino)purine cytokinins selected from the groupconsisting of the compounds of kinetin, benzyl adenine, isopentenyladenine, (6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),6-(3,3-dimethylallyl)aminopyrine, 6-(benzyl)aminopyrine,6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkylgroup has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and thosecompounds of Formula I,

in which R₁ is furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons,branched alkyl of 4, 5, or 6 carbons, (cyclohexyl)methyl,3,3-dimethylallyl, and 3-hydroxymethyl -3-methylallyl.
 47. (canceled)48. A method for treating neuronal degeneration or familial dysautonomiain a subject in need thereof, comprising administering to said subjectan effective amount of a composition that comprises one or morecytokinins, one or more cytokinins and one or more tocotrienols, or oneor more cytokinins and (−)-epigallocatechin gallate.
 49. The methodaccording to claim 48, wherein at least one of the one or moretocotrienols is tocotrienols selected from the group consisting ofα-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol.
 50. Themethod according to claim 48, wherein at least one of the one or moretocotrienols is δ-tocotrienol. 51-54. (canceled)
 55. The methodaccording to claims 46 or 48 wherein the subject is a mammal.
 56. Themethod according to claims 46 or 48 wherein the subject is a human. 57.The method according to claim 48, wherein at least one of the one ormore cytokinins selected from the group consisting of benzyladenine andkinetin.
 58. The method according to claim 48, wherein at least one ofthe one or more cytokinins is kinetin.
 59. A method of treating familialdysautonomia in a subject in need thereof, comprising administering tosaid subject a composition that comprises an effective concentration ofone or more 6-(substituted amino)purine cytokinins selected from thegroup consisting of the compounds of kinetin, benzyladenine, isopentenyladenine, (6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),6-(3,3-dimethylally)aminopyrine, 6-(benzyl)aminopyrine,6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkylgroup has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and thosecompounds of Formula I,

in which R₁ is furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons,branched alkyl of 4, 5, or 6 carbons, (cyclohexyl)methyl,3,3-dimethylallyl, and 3-hydroxymethyl-3-methylallyl.
 60. (canceled) 61.The method according to claim 59, wherein at least one of the one ormore tocotrienols selected from the group consisting of α-tocotrienol,β-tocotrienol, γ-tocotrienol, and δ-tocotrienol.
 62. The methodaccording to claim 59, wherein at least one of the one or moretocotrienols is δ-tocotrienol. 63-135. (canceled)
 136. The methodaccording to claim 59, wherein the subject is a mammal.
 137. The methodaccording to claim 59, wherein the subject is a human.